Transgenic non-human animals depleted in a mature lymphocytic cell-type

ABSTRACT

Transgenic mice having a phenotype characterized by the substantial depletion of a mature lymphocytic cell type otherwise naturally occurring in the species from which the transgenic mouse is derived. The phenotype is conferred in the transgenic mouse by a transgene contained in at least the precursor stem cell of the lymphocytic cell type which is depleted. The transgene comprised is a DNA sequence encoding a lymphatic polypeptide variant which inhibits maturation of the lymphocytic cell type.

This is a continuation Ser. No. 08/454,034, filed May 30, 1995 now U.S.Pat. No. 5,591,669, which is a continuation Ser. No. 07/919,936, filedJul. 27, 1992, now U.S. Pat. No. 5,434,340, which is a continuation ofSer. No. 07/280,218, filed Dec. 5, 1998, now U.S. Pat. No. 5,175,384,the disclosure of which is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to transgenic non-human animals whereinthe mature form of at least one lymphocytic cell type is substantiallydepleted. More particularly, the invention relates to transgenic micewherein mature T cells or plasma cells are depleted.

BACKGROUND OF THE INVENTION

The immune response is a complex defense system that is able torecognize and kill invading organisms such as bacteria, viruses, fungiand possibly also some types of tumor cells. The most characteristicaspects of the immune system are the specific recognition of antigens,the ability to discriminate between self and non-self antigens and amemory-like potential that enables a fast and specific reaction topreviously encountered antigens. The vertebrate immune system reacts toforeign antigens with a cascade of molecular and cellular events thatultimately results in the humoral and cell-mediated immune response.

The major pathway of the immune defense commences with the trapping ofthe antigen by accessory cells such as dendritic cells or macrophagesand subsequent concentration in lymphoid organs. There, the accessorycells present the antigen on their cell surface to subclasses of T cellsclassified as mature T helper cells. Upon specific recognition of theprocessed antigen the mature T helper cells can be triggered to becomeactivated T helper cells. The activated T helper cells regulate both thehumoral immune response by inducing the differentiation of mature Bcells to antibody producing plasma cells and control the cell-mediatedimmune response by activation of mature cytotoxic T cells.

Naturally occurring processes sometimes result in the modulation ofimmune system cell types. Acquired immunodeficiency syndrome (AIDS) is adevastating infectious disease of the adult immune system whichsignificantly affects cell-mediated immunity. This disease is manifestedby profound lymphopenia which appears to be the result of a loss ofT-lymphocytes which have the helper/inducer phenotype T4 as defined bythe monoclonal antibody OKT4 (Fauci, A., et al. (1984) Annals. Int. Med.100, 92). Other clinical manifestations include opportunisticinfections, predominantly Pneumocystis carinii pneumonia, and Karposi'ssarcoma. Other disease states include Severe Combined Immuno DeficiencySyndrome (SCID) wherein T, B or both cell types may be depleted inhumans. It is the existence of diseases affecting the immune system,such as AIDS and SCID, which has created the need for animal modelsystems to study the epitology and potential treatment of such diseasestates.

T lymphocytes recognize antigen in the context of self MajorHistocompatibility Complex (MHC) molecules by means of the T cellreceptor (TCR) expressed on their cell surface. The TCR is a disulfidelinked heterodimer noncovalently associated with the CD3 complex(Allison, J. P., et al. (1987) Ann. Rev. Immunol. 5, 503). Most T cellscarry TCRs consisting of α and β glycoproteins. T cells use mechanismsto generate diversity in their receptor molecules similar to thoseoperating in B cells (Kronenberg, M., et al. (1986) Ann. Rev. Immunol.4, 529; Tonegawa. S (1983) Nature 302, 575). Like the immunoglobulin(Ig) genes, the TCR genes are composed of segments which rearrangeduring T cell development. TCR and Ig polypeptides consist of aminoterminal variable and carboxy terminal constant regions. The variableregion is responsible for the specific recognition of antigen, whereasthe C region functions in membrane anchoring and in transmitting of thesignal that the receptor is occupied, from the outside to the inside ofthe cell. The variable region of the Ig heavy chain and the TCR β chainis encoded by three gene segments, the variable (V), diversity (D) andjoining (J) segments. The Ig light chain and the TCR α chain containvariable regions encoded by V and J segments only.

The V, D and J segments are present in multiple copies in germline DNA.The diversity in the variable region is generated by random joining ofone member of each segment family. Fusion of gene segments isaccompanied by insertion of several nucleotides. This N-region insertionlargely contributes to the diversity, particularly of the TCR variableregions (Davis and Bjorkman (1986) Nature 334, 395), but also impliesthat variable gene segments are often not functionally rearranged. Therearrangement of gene segments generally occurs at both alleles.However, T and B cells express only one TCR or Ig respectively and twofunctionally rearranged genes within one cell have never been found.This phenomenon is known as allelic exclusion.

During B cell development the rearrangement process starts at both heavychain gene alleles. First a D segment is fused to a J segment followedby ligation of a V segment to the DJ join. If the VDJ joining isproductive, further rearrangement of the other heavy chain allele isblocked, whereas rearrangement of the light chain loci is induced (Reth,M., et al. (1985) Nature 317, 353).

In both B and T cells, partially (DJ) and completely (VDJ) rearrangedgenes reportedly are transcribed giving rise to two differently sizedRNA molecules (Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339;Born, W., et al. (1987) TIG 3, 132). In B cells the DJ transcripts canbe translated into a Dμ-chain, a truncated form of the Igμ heavy chainthat lacks a V segment derived sequence. In general, the Dμ-chain ispresent in minor amounts, if at all. However, in one subclone (P4-11) ofthe 300-19 cell line, a transformed pre-B cell line which differentiatesin vitro to Ig producing B cells, the expression of the Dμ-chain isreportedly very high (Reth, M., et al. (1985) Nature 317, 353). Thisreference also reports that the heavy chain gene alleles in the P4-11clone are blocked at the DJ rearrangement stage in cell culture and thatsuch cells show a very high frequency of light chain generearrangements. This has led to the suggestion that the Dμ proteincontains some of the regulatory determinales necessary for gene assembly(Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339, 356).

Transgenic mice containing functionally rearranged Ig genes reportedlyhave been used in studying several aspects of Ig gene expression, e.g.tissue specific expression, the mechanism of segment rearrangement,allelic exclusion and repertoire development (Storb, U. (1987) Ann. Rev.Immunol, 5, 151). It has also been reported that the transgenic heavychain polypeptide only inhibits the complete VDJ rearrangement ofendogenous heavy chain genes if it contains a transmembrane domain(Storb, 1987; Iglesias, A., et al. (1987) Nature 330, 482; Nussenzweig,M., et al. (1987) Science 236, 816; Nussenzweig, M., et al. (1988) J.Exp. Med. 167, 1969).

Recently, the inventors reported that functionally rearranged TCRβ genescan be appropriately expressed in transgenic mice (Krimpenfort, P., etal. (1988) EMBO 7, 745). This functional TCRβ chain gene preventsexpression of endogenous β genes by inhibiting complete VDJ joining(Uematsu, Y., et al. (1988) Cell 52, 831).

Two different types of T cells are involved in antigen recognitionwithin the self MHC context. Mature T helper cells (CD4⁺ CD8⁻) recognizeantigen in the context of class II MHC molecules, whereas cytotoxic Tcells (CD4⁻ CD8⁺) recognize antigen in the context of class IMHCdeterminants (Swain, S. L. (1983) Immun. Rev. 74, 129-142; Dialynas, P.D., et al. (1983) Immun. Rev. 74, 29-56). It has been reported thatclass II- specific CD4⁺ CD8⁻ helper T cells (also referred to as T4cells) fail to develop in mice neonatally treated with anti-class II MHCmonoclonal antibody (Kruisbeek, A. M., et al. (1983) J. Exp. Med. 157,1932-1946; Kruisbeek, A. M., et al. (1985) J. Exp. Med. 161, 1029-1047).Similarly, it has recently been reported that mice chronically treatedwith anti-class I MHC monoclonal antibody from birth have asignificantly reduced population of CD4⁻ CD8⁺ cells and cytotoxic T cellprecursors (Marusic-Galesic, S., et al. (1988) Nature 333, 180-183).Although selected T cell populations apparently can be produced by suchmethods, continuous administration of antibody is required which oftenresults in adverse side effects in such mice.

A different strategy to deplete specific cell lines has recently beenidentified wherein specific cell destruction is induced byadministration of a toxic metabolite. Specifically, transgenic micereportedly were produced containing a Herpes Simplex Virus ThymidineKinase (HSV-TK) transgene fused to the Ig promoter/enhancer. Transgeniccells that express the HSV-TK are not affected. However, uponadministration of a nucleoside analog that can be phosphorylated by thetransgenic HSV-TK gene, replicating cells expressing the HSV-TK gene arekilled (Heyman, et al. (1988) UCLA Symposia on Molecular and CellularBiology, 73, 199.

Another approach to depletion of specific cell types has been reportedusing tissue specific expression of a bacterial toxin. Specifically,mice carrying an elastase/diptheria toxin A (DT-A) fusion gene lacked anormal pancreas (Palmeter, et al. (1987) Cell 50, 435). In addition, ithas been reported that microphtalmia in transgenic mice resulted fromthe introduction of the DT-A gene fused to the α2-crystallin promoter(Bretman, et al. (1987) Science 238, 1563).

Transgenic mice reportedly have also been constructed that express anαβTCR in a large fraction in their T cells which is specific for a minorhistocompatibility antigen (H-Y) present on male, but not female, cells(Kisielow, P., et al. (1988) Nature 333, 742-746). This very recentreference reports that cells containing the TCR for the H-Y antigen werefrequent in female but not in male transgenic offspring. The αβ TCR insuch trasgenic mice apparently contains all the segments and regionsrequired for a functional TCR.

The references discussed above are provided solely for the disclosureprior to the filing date of the present application and nothing hereinis to be construed as an admission that the inventors are not entitledto antedate such disclosures by virtue of prior invention.

Given the state of the art, it is apparent that a need exists for animalmodel systems to study diseases which effect the immune system includinginfectious diseases such as AIDS. Accordingly, it is an object herein toprovide transgenic non-human animals and methods for making the samewhich have a phenotype characterized by the substantial depletion of amature lymphocytic cell type otherwise naturally occurring in thespecies from which the transgenic is derived.

It is also an object herein to provide transgenic non-human animalssubstantially depleted in mature T cells or plasma cells.

It is a further object herein to provide transgenic mice substantiallydepleted in mature T cells or plasma cells.

Still further, it is an object herein to provide transgenes capable ofproducing such transgenic non-human animals.

Further, it is an object herein to provide methods for producingtransgenic non-human animal having at least one of the above identifiedphenotypes.

SUMMARY OF THE INVENTION

The invention is based on the discovery that transgenic non-humananimals depleted in a lymphatic cell type can be produced by disruptingthe expression of a functional lymphocytic polypeptide required formaturation of the lymphocytic cell type. This lymphocytic polypeptide isotherwise expressed by the non-human animal from which the transgenicanimal is derived.

In one aspect, the invention provides transgenic non-human animalshaving a phenotype characterized by the substantial depletion of amature lymphocytic cell type otherwise naturally occurring in thespecies from which the transgenic animal is derived. This phenotype isconferred in the transgenic animal by a transgene contained in at leastthe precursor stem cell of the lymphocytic cell type which is depleted.The transgene comprises a DNA sequence encoding a lymphatic polypeptidevariant which inhibits formation of the depleted lymphocytic cell type.Generally, such inhibition occurs when the lymphatic polypeptide variantis expressed in a precursor to the lymphocytic cell type.

In those cases where the lymphatic polypeptide variant is expressed, thevariant is believed to be capable of suppressing expression of at leastone set of cognate endogenous alleles normally expressed duringdifferentiation of the precursor stem cell to the mature lymphocyticcell type. The lymphatic polypeptide variant, however, lacks afunctional domain necessary for maturation of the lymphocytic cell typewhich would otherwise be provided by either or both of the suppressedendogenous alleles.

Within the context of transgenic animals deficient in T cells, thetransgene encodes a lymphatic polypeptide variant comprising a portionof a TCRβ chain. The transgene encoding the TCRβ variant chain typicallyretains sequences encoding at least the transmembrane sequence found inthe C region of a naturally occurring TCRβ chain. This sequence may beoperably linked to an appropriate signal sequence. It lacks, however,sequences encoding all or part of the variable region. The C regioncontained by such a lymphatic polypeptide variant is capable ofsuppressing the expression of endogenous TCR alleles thereby preventingthe membrane expression of functional heterodimeric TCRs. Normal T cellmaturation is thereby abrogated.

In the case of non-human transgenic animals substantially depleted inantibody secreting plasma cells, the transgene similarly encodes alymphatic polypeptide variant containing at least the transmembranesequence of the C region of the Ig heavy chain. A signal sequence mayalso be operably linked to the transgene encoding the lymphaticpolypeptide variant.

The invention also includes transgenes comprising a DNA sequenceencoding a lymphatic polypeptide variant.

Further, the invention includes a method for producing a transgenicnon-human animal substantially depleted in a mature lymphocytic celltype. The method comprises introducing a transgene into an embryonaltarget cell. The transgene encodes a lymphatic polypeptide variant andis capable of inhibiting formation of a mature lymphocytic cell type.The thus transformed transgenic embryonal target cell is thereaftertransplanted into a recipient female parent from which offspring havingthe desired phenotype are identified.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the αβ T cell receptor.

FIG. 1B depicts the αβ T cell receptor variant containing a deletion ofthe variable region of the β chain.

FIG. 2 depicts the genomic organization of the T cell receptor loci inmouse.

FIG. 3A and 3B show the structure of the T cell receptor β transgeneincluding nucleotide sequence of the variable region and restriction mapof clone cos HY9-1.14-5.

FIG. 4 is a Southern blot analysis of offspring mice derived from across of the female transgenic mouse 93 with C57L males.

FIGS. 5A-5F demonstrates the surface expression of F23.1-positive βchains on T cells from transgenic and normal mice.

FIGS. 6A and 6B demonstrate the tissue specificity of transgenetranscription.

FIG. 7 is a gel electrophoretic analysis of immuno percipitated β chainsfrom transgenic T lymphocytes before and after preclearing with F23.1monoclonal antibodies.

FIGS. 8A-8I demonstrates the modulation of surface expression of CD3molecules on various T cells by Vβ8-specific F23.1 monoclonalantibodies.

FIGS. 9A-9C demonstrates the inhibition of cytllytic activity oftransgenic T cells by Vβ8-specific F23.1 monoclonal antibodies.

FIGS. 10A, 10B, and 10C demonstrate that endogenous β genes intransgenic mouse 93.2 show predominantly partial Dβ1-Jβ rearrangements.

FIGS. 11A, 11B-1 and 11B-2 and 12 depict the construction of ΔV-TCRβ andΔV.sub.▪ -TCRβ transgenes.

FIGS. 13A and 13B demonstrates the expression of the ΔV.sub.▪ -TCRβtransgene.

FIGS. 14A-14H is a surface marker staining profile of splenocytes fromtransgenic mouse #1670 and a control mouse.

FIGS. 15A, 15B and 15C depict the amino acid and known DNA sequence fortransgenes TCRβ and ΔV-TCRβ based on partial CDNA and genomic sequencedata.

DETAILED DESCRIPTION OF THE DISCLOSURE

The "non-human animals" of the invention comprise any non-human animalhaving an immune system capable of producing a humoral and/orcell-mediated immune response. Such non-human animals includevertebrates such as rodents, non-human primates, sheep, dog, cow,amphibians, reptiles, etc. Preferred non-human animals are selected fromthe rodent family including rat and mouse, most preferably mouse.

The "transgenic non-human animals" of the invention are produced byintroducing "transgenes" into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for micro-injection. In the mouse, the male pronucleus reachesthe size of approximately 20 micrometers in diameter which allowsreproducible injection of 1-2 pl of DNA solution. The use of zygotes asa target for gene transfer has a major advantage in that in most casesthe injected DNA will be incorporated into the host gene before thefirst cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. U.S.A.82, 4438-4442). As a consequence, all cells of the transgenic non-humananimal will carry the incorporated transgene. This will in general alsobe reflected in the efficient transmission of the transgene to offspringof the founder since 50% of the germ cells will harbor the transgene.Microinjection of zygotes is the preferred method for incorporatingtransgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad.Sci U.S.A. 73, 1260-1264). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner, et al. (1985) Proc. Natl.Acad. Sci. U.S.A. 82, 6927-6931; Van der Putten, et al. (1985) Proc.Natl. Acad. Sci U.S.A. 82, 6148-6152). Transfection is easily andefficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (Van der Putten, supra; Stewart, et al. (1987)EMBO J. 6, 383-388). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner, D., et al. (1982) Nature 298, 623-628). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic non-humananimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner, D.et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans, M. J., et al. (1981)Nature 292, 154-156; Bradley, M. O., et al. (1984) Nature 309, 255-258;Gossler, et al. (1986) Proc. Natl. Acad. Sci U.S.A. 83, 9065-9069; andRobertson, et al. (1986) Nature 322, 445-448). Transgenes can beefficiently introduced into the ES cells by DNA transfection or byretrovirus-mediated transduction. Such transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells thereafter colonize the embryo and contribute to the germ line ofthe resulting chimeric animal. For review see Jaenisch, R. (1988)Science 240, 1468-1474.

As used herein, a "transgene" is a DNA sequence introduced into thegermline of a non-human animal by way of human intervention such as byway of the above described methods. The transgenes of the inventioninclude DNA sequences which are capable of suppressing cognateendogenouos alleles. Further, such transgenes are capable of eitherfacilitating or inhibiting the maturation of a lymphatic cell type. Suchtransgenes comprise DNA sequences encoding either a "lymphaticpolypeptide" or "lymphatic polypeptide variant" which may be expressedin a transgenic non-human animal.

A lymphatic polypeptide corresponds to a naturally occurring polypeptideexpressed selectively by lymphatic tissue and includes the wide varietyof polypeptide chains associated with immunoglobulin production byplasma cells and TCRs produced by mature T cells. Such lymphaticpolypeptides are produced by mature lymphocytic cell types afterdifferentiation. Such differentiation involves the functionalrearrangement of numerous gene regions and segments to form the DNAsequence encoding the naturally occurring lymphatic polypeptide(described in more detail hereinafter).

The term "cognate endogenous alleles" refers to alleles in the genome ofthe transgenic non-human animal which are closely related to thetransgene introduced therein. Thus, for example, the alleles involved inTCRβ chain production in mouse T cells are cognate endogenous alleleswhich may be suppressed by a transgene comprising a DNA sequenceencoding a TCRβ chain from mouse or other species. Further, such cognateendogenous alleles may include the γδ alleles involved in γδ TCRproduction.

The transgene encoding a lymphatic polypeptide, in addition tosuppressing cognate endogenous alleles, is also capable of facilitatingthe maturation of the particular lymphocytic cell type which wouldnormally express the cognate endogenous alleles if the lymphaticpolypeptide is expressed. Thus, transgenic mice containing a transgeneencoding a functionally rearranged βTCR chain contain mature Tlymphocytes which express αβTCRs containing β chains encoded by the TCRβtransgene.

Transgenes encoding lymphatic polypeptide variants, however, suppresscognate endogenous alleles and inhibit the maturation of the lymphocyticcell type which normally expresses such cognate endogenous alleles. Theparameters which define the lymphatic polypeptide variant may be definedstructurally and functionally.

As indicated in the Examples, a functionally rearranged mouse TCR fusionβ gene which is expressed in a transgenic mouse, suppresses theexpression of the cognate endogenous alleles encoding the β chain TCRgene. See also Uematsu, Y., et al. (1988) Cell 52, 831-841. Thesuppression of such expression apparently results from the blocking ofthe rearrangement process of the endogenous β genes duringdifferentiation to the mature T lymphocyte. Partial D-J rearrangementsare found in such transgenic animals while complete VDJ rearrangementsare not detected. Such transgenic mice, however, do produce functionalmature T cells. The αβ TCRs on such trangenic mice are homogeneous withrespect to the β chain encoded by the transgene. They are, however,fully functional, at least in a variety of allogenic responses,presumably due to the diversity produced in such receptors by way ofendogenous α chain rearrangements.

When, however, transgenic mice are produced containing a functionallyrearranged TCR β gene which has had approximately 90% of the variableregion deleted (ΔTCRβ), such mice failed to form a functional thymus andsubstantial T cell depletion was observed. Thus, disruption of theexpression of functional TCRβ chains is sufficient to cause T celldepletion. The mechanism for this T cell depletion is not known.However, it is assumed that the TCR β gene encodes a lymphaticpolypeptide having at least two functional domains related to themechanism of T cell depletion when transgenes containing variable regiondeletions are used. One domain functions to suppress the expression ofcognate endogenous alleles (such as the TCRβ chain alleles) presumablyby a mechanism analogous to allelic exclusion observed during normaldifferentiation. The second functional domain is required for T cellmaturation. In the case of the complete TCR β gene both functionaldomains are present in the trangenic TCR β chain as evidenced by thesuppression of endogenous β alleles and the normal maturation of Tlymphocytes. However, in the case of the transgene ΔV-TCRβ (containing adeletion of approximately 90% of the variable region), the transgenicΔV-TCRβ polypeptide contains only a first functional domain capable ofsuppressing endogenous β chain alleles. A sufficient portion of thesecond functional domain is deleted such that T cell maturation isinhibited. See FIG. 1B.

Thus, structurally, the transgene, in one aspect of the invention,encodes a lymphatic polypeptide variant comprising a lymphaticpolypeptide wherein all or part of the variable region is deleted.Preferably, at least part of the V segment of the V region is deleted.However, the deleted sequences may also include part of the D and/or Jsegment of the variable region of the lymphatic polypeptide.

The transgene encoding the lymphatic polypeptide variant also can bedefined functionally. This definition is based on the two functionaldomains present in the naturally occurring lymphatic polypeptide. Byanalogy to the TCRβ chain, the first functional domain is capable ofsuppressing the expression of cognate endogenous alleles whereas thesecond functional domain is capable of inducing the maturation of aparticular lymphocytic cell type. The lymphatic polypeptide variantcontains the first functional domain but lacks the second. Althoughdefined functionally, such domains can be structurally ascertained byway of the methodology disclosed in Examples 1 and 2 herein. Forexample, the first functional domain involved in the suppression ofcognate endogenous alleles may be ascertained by generating a family oftransgenes encoding specific deletions of portions of the lymphaticpolypeptide. Transgenic mouse lines, containing each of thesetransgenes, for example, may then be analyzed to ascertain thesuppression of endogenous alleles as described in Example 1. Thosetransgenes containing deletions which do not induce suppression of theendogenous alleles define the functional domain responsible forsuppression of endogenous expression.

Once such structural domains are defined, the second functional domainmay be defined structurally by generating a series of transgeneconstructs containing deletions of various portions of the lymphaticpolypeptide not defining the first functional domain. Thus, sequentialdeletions (i.e., deletions of about 10-30 base pairs through, forexample, the variable region) or progressive deletions (i.e., a set ofdeletions comprising deletion of approximately 10-20 base pairs to, forexample, all of the DNA sequence encoding the variable region) can beused in conjunction with the methods of Example 2 to define the secondfunctional domain required for lymphocyte maturation. Thus, lymphaticpolypeptide variants and the transgenes encoding the same can be readilydefined by such methods.

As indicated, the lymphatic polypeptide variant encoded by the transgenein one embodiment of the invention should be expressed in a precursor ofthe mature lymphocyte to be depleted in the transgenic animal. To beexpressed, the transgene DNA sequence must contain regulatory sequencesnecessary for transcription of the transgene in the precursor cell typein the transgenic animal. Further, various regulatory sequencesnecessary for translation and if necessary processing of the mRNAtranscripts encoded by the transgene are required. Such regulatorysequences include promoter sequences to control transcription, ribosomebinding sites to facilitate translation, splice acceptor and splicedonor sequences for intron excision, polyadenylation sequences,transcription and translational termination signal and enhancersequences which facilitate the efficient expression of the lymphaticpolypeptide variant. In most cases, a secretory signal sequence willalso be included to facilitate the association of the lymphaticpolypeptide variant with the membrane of the cell expressing thetransgenic polypeptide. Transgenes therefore encode all of theregulatory sequences necessary to express the lymphatic polypeptide orlymphatic polypeptide variant encoded by the transgene. Of course, eachof the regulatory sequences and encoding sequences are operably linkedto produce a functional transgene capable of expressing the trangenicpolypeptides in the non-human transgenic animal.

"Operably linked" when describing the relationship between two DNA orpolypeptide sequences simply means that they are functionally related toeach other. For example, a signal or leader sequence is operably linkedto a peptide if it functions as a signal sequence participating in theinsertion of the immature form of the protein into a cell membrane.Similarly, a promoter is operably linked to a coding sequence if itcontrols the transcription of the sequence; a ribosome binding site asoperably linked to a coding sequence if it is positioned so as to permittranslation, etc.

Transgenes are derived, for example, from DNA sequences encoding atleast one polypeptide chain of a T cell receptor (TCR) or onepolypeptide chain of the immunoglobulin (Ig) molecule. Preferably, amodified for of the β or γ chain of the TCR and most preferably the βchain of the TCR is used as a transgene to inhibit the formation ofmature T cells. In the case of B cells, a derivative of the heavy chainof the Ig molecule is preferred to inhibit the formation of antibodyproducing plasma cells derived from B cells. Generally, such transgenesare derived by deleting from the DNA sequence encoding a functionallyrearranged β chain, γ chain or heavy chain polypeptide, all or part ofthe DNA sequence encoding the variable region of such molecules.Preferably all of the variable region is deleted although small segmentsof 5' sequences encoding an N-terminal portion of the V segment and 3'sequences, encoding the C-terminal portion of the J segment may beretained in the transgene. At the very least, all or part of thevariable segment should be deleted. Thus, transgenes generally compriseC regions of the β chain, γ chain or heavy chain polypeptides of TCR andIg molecules respectively but may also include additional sequencesencoding all or part of the J and D segments of the variable region.

Transgenic mice containing a transgene encoding a TCRβ chain having itsvariable regions deleted do not contain detectable T cells. Further,transgenic mice containing immunoglobulin heavy chain wherein all orpart of the variable region is deleted are expected to be incapable ofproducing plasma cells which secrete immunoglobulins. This is becauseduring B cell development, B cells rearrange their Ig heavy chain genesfirst. Once a functionally rearranged Ig heavy chain gene is generated,light chain rearrangement starts. This eventually will result in theproduction of complete IgM molecules containing two heavy and two lightchains. Such IgMs are not secreted since their C regions contain amembrane anchoring domain. During further develoment, the B cells switchthe use of the constant region to other constant regions that do notencode a transmembrane domain, e.g., IgG. When this switch occurs, theybecome plasma cells which secrete large amounts of specificimmunoglobulin. In order to develop into plasma cells, the IgM producingB cells must interact with other cells of the immune system via the IgMlocated on the B cell surface. Since the heavy chain of the IgM variantcontains the deletion of all or part of the variable region, transgenicnon-human animals containing a transgene encoding such a deleted heavychain should not be able to produce B cells which can interact with theimmune system to form the mature plasma cell type.

Alternatively, the transgene may comprise a DNA sequence encoding alymphatic polypeptide variant which is incapable of being expressed inthe precursor stem cell or latter precursor of the lymphocytic cell typeto be depleted. Such DNA sequences may contain one or more mutationsinvolving the insertion or deletion of one or more nucleotides that may,for example, result in a frame shift or nonsense mutation (e.g., stopcodon) that prevents all or part of the expression of the transgene. Thetransgene, however, has sufficient sequence homology with a cognateendogenous allele such that when introduced into an ES target cell itmay homologously recombine with such an allele in the ES cell to disruptits expression. See, e.g., Kirk and Capecchi (1978) Cell 51, 503-512.After identification and selection of ES cells containing the transgenein the targeted allele, a transgenic non-human animal may be produced bycolonizing an appropriate embryo with the selected ES cell.

In the case of T cells, the Cβ1 and Cβ2 alleles for the TCRβ chain andmost preferably the Cα allele for the TCRα chain are targeted forinsertion of a transgene which disrupts expression of the allele. Forexample, in the case of the Cα allele, once a genotype is identifiedcontaining a transgene disrupting Cα expression, cross-breeding can beused to produce transgenic animals homozygous for the Cα⁻ genotype. Suchhomozygotes should also be deficient in mature T cells since they areincapable of producing the functional αβTCR required for T cellmaturation.

A similar approach can be used to produce plasma cell deficienttransgenic animals. In that case the transgene is targeted to disruptthe expression of Cμ and Cδ portions of the IG heavy chain.

Transgenic non-human animals depleted in one or more mature lymphocyticcell types, such as transgenic mice depleted in mature T cells or plasmacells, have multiple uses. For example, T cell depleted mice do not havea cell-mediated immune response. They are therefore suitable for testingdrugs that interfere with cell-mediated immunity for side effects andcomplications, e.g., kidney damage, etc. Thus, antibiotics, anti-viraldrugs, antifungal agents and immunosuppressive drugs such ascyclosporine may be administered to such mice to ascertain theireffects.

In addition, such T cell deficient mice do not have T4 (helper) cellsused in the B cell humoral response. They can, therefore, be used fortesting the effects of passive immunization. Further, the lack of acell-mediated immune response renders such transgenic mice prone todevelop tumors since they are not protected by immuno-surveillance. Theytherefore can be used to test the carcinogenicity of various materials.In addition, materials may be tested in such animals which may conferprotection against the development of neoplasms.

Further, such T cell depleted mice can be used as model systems for AIDSand SCID involving the depletion of T cells.

Plasma cell deficient transgenic mice, in combination with T celldepleted mice, offer a good model system to study the effect of thedepletion of the humoral immune response, cell-mediated immune responseor both. Further, such plasma cell depleted and/or T cell depleted micecan be used as a model system to study SCID syndromes involving thedepletion of B, T and/or B and T cell types.

The genes encoding the various segments and regions which may be used inthe invention are well characterized. The TCRs associated with T cellsrepresent an enormous percent of clonally varying molecules with thesame basic structure. The TCR is a heterodimer of 90 kd consisting oftwo transmembrane polypeptides of 45 kd each connected by disulfidebridges (FIG. 1A) (Samuelson, et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80, 6972; Acuto, et al. (1983) Cell 34, 717; MacIntyre, et al.(1983) Cell 34, 737). For most T cells, the two polypeptides arereferred to as the α and β chain. Like the heavy and light chains of theimmunoglobulins, the α and β chains have variable (V) and constant (C)regions (Acuto, et al. (1983) supra; Kappler, et al. (1983) Cell 35,295). The V region is responsible for antigen recognition and the Cregion is involved in membrane anchoring and signal transmission. Asmall percentage of lymphoid T cells, however, have a different set ofTCRs comprising different polypeptide chains referred to as γ and δchains (Borst, et al. (1987) Nature 325, 683; Brenner, et al. (1987)Nature 325, 689; Bank, et al. (1986) Nature 322, 179; Pardoll, et al.(1987) Nature 326, 79).

Using subtractive hybridization procedures, cDNA clones encoding the TCRpolypeptide chains have been isolated (Hendrick, et al. (1984) Nature308, 149; Hendrick, et al. (1984) Nature 308, 153; Yanagi, et al. (1984)Nature 308, 145; Saito, et al. (1987) Nature 325, 125; Chien, et al.(1984) Nature 312, 314). Sequence analysis of these cDNA clones revealthe complete primary sequence of the TCR polypeptides. The TCRpolypeptides are all similar to each other and to the immunoglobulinpolypeptides. For review see Davis and Bjorkman (1988) supra.; andKronenberg, et al. (1986) Ann. Rev. Immunol. 4, 529). The variableregion of the TCR chains is composed of a variable region (V) and aconstant region (C). The variable region of the TCR chains is furthersubdivided into a variable (V) and joining (J) segments. In addition,the variable region of the β and δ chains also contains a diversity (D)segment interposed between the V and J segments. At similar positions asin the immunoglobulins, hypervariable regions are present in thevariable region of the TCR. Two hypervariable regions are encoded in theV region whereas one is represented by the junction between the V and Jor V, D and J segments. The constant region of the TCR chains iscomposed of four functional regions often encoded by different exons(Davis and Bjorkoran (1988) supra.). The TCR C regions show typicalimmunoglobulin-like characteristics such as the presence of cystineresidues involved in the linking of two β sheets which probably foldinto immunoglobulin-like domains. All TCR chains have a hydrophobictransmembrane region in which a highly conserved lysine residue ispresent.

The availability of TCR cDNAs permits an analysis of the genomicorganization of the murine and human TCR genes. The TCR genes show asegmental organization similar to the immunoglobulin genes. FIG. 2 showsa schematic representation of the four TCR gene loci. In the β chaingene locus, two nearly identical Cβ regions are tandemly arranged, eachpreceded by one D and six J segments (Kronenberg, et al. (1986), supra;Davis, M. (1985) Rev. Immunol. 3, 537). The β locus also containsapproximately 20 to 30 V segments (Barth, et al. (1985) Nature 316, 517;Behlkey, et al. (1985) Science 229, 566), one of which is located 3' tothe C regions in opposite orientation (Malissen, et al. (1986) Nature319, 28). The γ locus is less diverse, containing three J.sub.γ -C.sub.γregions and only a limited number of V segments (Hayday, et al. (1985)Cell 40, 259; Chien, et al. (1987) Nature 330, 722). The α and δ locioverlap each other in that many of the δ coding segments are locatedwithin the segments of the α gene (Chien, et al., supra; Elliott, et al.(1988) Nature 331, 627). As a consequence, the δ segments are deleted inmost αβ bearing T cells (Lindsten, et al. (1987) J. Exp. Med. 166, 761).Thus, the α and γ chain genes are encoded by variable and joiningsegment and constant regions whereas the β and δ chain genes containvariable, diversity and joining segment and a constant region. Duringsomantic development of the T cell, a functional TCR gene is formed byrearrangement of these segments and regions. This process is the basisfor T cell receptor diversity. The following strategies have beenpostulated for T cell diversification: (1) multiple germline V and Jsegments (Kronenberg, et al. (1986) supra; (2) D segments that can betranslated in all three reading frames in the case of β and δ genes(Goverman, et al. (1985) Cell 40, 859; Elliott, et al. (1988), Nature331, 627); (3) combinatorial joining of V, D and J segments; (4) therandom addition of nucleotides between the V, D and J segments (N-regionaddition) (Davis and Bjorkman (1988) supra); (5) the flexibility ofseveral V segments with respect to the position of their 3' joiningpoints (Davis and Bjorkman, supra); and (6) the combinatorial joining ofTCR chains.

As shown schematically in FIG. 2, the encoding segments for the TCRgenes are scattered over large arrays of chromosomal DNA. Like theimmunoglobulin genes, specific V, D and J segments are fused together togenerate a complete V coding region next to a C region. In B and Tcells, the rearrangements are mediated by similar sequences flanking thesegments to be fused (Akira (1987) Science 238, 1134; Yancopoulos, etal. (1986) supra.). These sequences consist of conserved heptamer andmonomer stretches spaced by 12 or 23 nucleotides. Depending on theorientation of the segments being joining, looping out/deletion orinversion of large genomic DNA can occur (Fujomoto (1987) Nature 327,242; Okazaki, et al. (1987) Cell 49, 477; Mallisen, B., et al. (1986)supra). B and T cells probably use the same machinery for the assemblyof Ig and TCR since B cells rearrange transfected TCR segments in thesame way as transfected Ig gene segments (Yancopoulos, et al. (1986)supra). The TCR β, γ and δ genes are rearranged and transcribed first,followed by the TCR α gene (Chien, et al. (1987) supra; Pardoll, et al.(1987) Nature 326, 79; Raulet, et al. (1985) Nature 312, 36; Samelson,et al. (1985) Nature 315, 765; Snodgrass, et al. (1985) Nature 315,232).

The regulation of immunoglobulin gene assembly and expression isintrinsicly related to the progression of B-cell precursors to the Bcell differentiation stage. For a review of the mechanism of Ig variableregion gene assembly and regulatory mechanisms to control genomicrearrangements, see Yancopoulos, et al. (1986) supra. Ig gene assemblyin B cells closely parallels that for TCR assembly in T cells. Inaddition, a number of transgenic mice containing a number ofimmunoglobulin genes have been prepared to further study the mechanismof Ig expression, allelic exclusion and rearrangement mechanisms (Storb(1987) Ann. Rev. Immunol. 5, 151-174).

Thus, the αβ and γδ TCRs of T cells and the heavy and light chains of Igmolecules in B cells have been well characterized. As indicated, thetransgenes of the present invention are derived form such DNA sequences,preferably those encoding the β and γ chains of the T cell receptors,most preferably the β chain, and the heavy (H) chain of the Ig moleculefrom B cells. Such DNA may be obtained from the genome of somatic cellsand cloned by well established technology. Such cloned DNA sequences maythereafter be further manipulated by recombinant techniques to constructthe transgenes of the present invention. However, a more efficient andpreferred methodology is to clone a functionally rearranged β or γ TCRgene or a functionally rearranged heavy Ig gene. Such clonedfunctionally rearranged genes generally will have all necessaryregulatory and secretory sequences for expression of the functionallyrearranged gene in the B or T lymphocytes of the animal from which thesequence is derived, including introns which are necessary for theefficient expression of transgenes (Brinster, et al. (1988) Proc. Natl.Acad. Sci. U.S.A. 85, 836). Such DNA sequences can be derived bygenerating cultures of T or B cells from mature animals or animals whichhave been challenged with an antigen. The DNA from one or more suchclones may be used to generate a genomic DNA library in hosts such asλgt11 and EMBL 3 following published procedures (Young, R. A., et al.(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1194-1198; Frischauf, et al.(1983) J. Mol. Biol. 170, 827-842). Such libraries may then be screenedwith appropriate probes to particular regions or segments of the βand/or γ genes for TCRs or the heavy gene for Ig molecules depending onthe source of the genomic DNA. Such probes preferable are specific for apart of one of the C regions of the molecule of interest. cDNA clonesfrom the same cellular clone may also be generated and identified bysimilar probes. The sequences of such cDNA clones facilitates theidentification of the coding sequence of the genomic clone therebypermitting the identification of putative introns and regulatorysequences.

If a particular genomic clone does not contain the entire coding regionfor the functionally rearranged chain or if it does not containregulatory or enhancer regions typically located in 5' or 3' flankingregions, that DNA sequence may be recombined with other clones toprovide such sequences. Thus, for example, a genomic clone from the Tcell clone B6.2.16 (described in more detail in the Examples) hasflanking sequences comprising 2 kb 5' and 2 kb 3' from the coding regionfor the mouse TCR β gene. This DNA sequence is inactive in transgenicmice because it lacks a 550 base pair enhancer sequence located 5 kbdownstream from the Cβ2 locus (Krimpenfort, P., et al. (1988) EMBO J. 7,745-750). A fusion construct was therefore prepared containing a largerextension of the 3' end. A cosmid clone derived from BALB7c liver DNAcontaining the Cβ2 and downstream region was used to generate thisfusion construct which is active in transgenic mice (Krimpenfort, P., etal. (1988) supra). Similar methods may be used to generate fusion genescontaining all the necessary DNA sequences for regulation of theexpression of the DNA sequences encoding the particular polypeptidechain of interest.

The DNA sequences from which the transgene is derived are preferablyobtained from the functionally rearranged genome of the same species ofanimal into which the transgene will be introduced. Thus, functionallyrearranged β and γ genes from mouse TCR are preferred for makingtransgenes for use in transgenic mice. The invention, however, is notlimited to transgenes derived from the same species of animal used toform transgenic non-human animals. It has recently been shown that twoindependent lines of transgenic mice containing either the human heavychain of HLA class I antigen or the light (β₂ -microglobulin) chain ofhuman HLA class I antigen can be crossed to produce a functional HLAclass I antigen which is biochemically indistinguishable from the sameantigen expressed on human cells (Krimpenfort, P., et al. (1987) EMBO J.6, 1673-1676. Other examples of heterologous transgenic expressioninclude the heterologous regulatory and structural sequences disclosedin EPO Publication No. 0247494 and PCT Publication No. WO88/00239. Thus,DNA sequences from species different from that of the transgenic animal("heterologous DNA") may be used to form the transgenic non-humananimals of the present invention. Such heterologous sequences includeregulatory and secretory sequences as well as structural DNA sequenceswhich when modified encode heterologous lymphatic polypeptide variantscapable of inhibiting the formation of a mature lymphocytic cell type.The only limitation on the use of such heterologous sequences isfunctional. The heterologous regulatory sequences must be utilized bythe transgenic animal to efficiently express sufficient amounts of thelymphatic polypeptide variant such that it is able to inhibit theformation of a mature lymphocytic cell type. Further, the heterologouslymphatic polypeptide variant when properly expressed in the transgenicanimal must be capable of producing the desired depletion of alymphocytic cell type. Thus, by analogy to expression of human HLAantigens on the cell surface of transgenic mice, a functionallyrearranged human TCRβ gene containing a variable region deletion can beused to express a human β TCR variant capable of suppressing theformation of mature T cells in transgenic mice. Further, it should bepossible to mix homologous and heterologous DNA sequences (e.g.,homologous regulators with heterologous structural genes and vice versa)to produce functional transgenes which may be used to practice theinvention. Alternatively, heterologous DNA sequences encoding lymphaticpolypeptide variant must be capable of inhibiting the expression offunctional lymphatic polypeptide required for the maturation of alymphocytic cell type by disrupting the expression of cognate endogenousalleles.

The following is presented by way of example and is not to be construedas a limitation on the scope of the invention. Further, all referencesreferred to herein are expressly incorporated by reference.

EXAMPLE 1 Suppression of Cognate Endogenous Alleles of TCRβ

This example describes the construction of a transgene which whenexpressed is capable of suppressing the expression of cognate endogenousalleles for the TCRβ chain. When introduced into a mouse, the transgenicmouse so produced expresses TCR receptors substantially containing onlythe TCRβ chain encoded by the transgene.

Establishment of the Male-Specific B6.2.16 Clone

C57BL/6 female mice were immunized with 10⁷ male spleen cells injectedintraperitoneally. After 14 days, 2×10⁷ female spleen cells werecultured with 2×10⁷ x-irradiated male cells in 8 ml of culture media.After 12 days, 10⁶ viable female responder cells were restimulated with10⁷ x-irradiated male cells in 1 ml of medium containing interleukin-2(IL-2). Cells were cloned by culturing 0.3 cells with stimulators asdescribed below. Clones were tested for cytolytic activity as describedbelow, and clone B6.2.16 was selected after surface staining of variousclones with the F23.1 antibody (von Boehmer and Haas, (1986) Meth.Enzymol. 132, 467-477; Staer, et al. (1985) J. Immunol. 134, 3994-4000)which binds to all three members of the Vβ8 family (Behlke, et al.(1982) J. Exp. Med. 165, 257-267).

Isolation and Characterization of β Chain cDNA and Genomic Clones

Total RNA and high molecular weight DNA were isolated from cytotoxic Tcell clone B6.2.16 and used to construct cDNA and genomic DNA librariesin λgt11 and EMBL3, respectively, following published procedures (Youngand Davis (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1194-1198; Frischauf,et al. (1983) J. Mol. Biol. 170, 827-842). Both libraries were screenedwith a Cβ probe corresponding to the sequence of the Cβ2 region reportedby Mallisen, et al. (1984) Cell 37, 1101. Positive clones werecharacterized by mapping with restriction endonucleases andhybridization with probes specific for the Dβ1 gene segment, 1 1.4 kpPst1 fragment entending 1.25 kb 5' to the Dβ1 gene segment (Siu, et al.(1984) Nature 311, 344-350), and Jβ gene segment. A full-length cDNAclone and genomic clones, λ8 for the functional allele and λ3 for thenonfunctional allele, were subcloned into M13mp18 for DNA sequencedetermination. Sequencing primers were the 17-mer universal M13 primer(Amersham) and 15- to 17-mer oligonucleotides synthesized to extend DNAsequence information beyond what was determined with M13 primers.

DNA sequence determination of the cDNA clone (FIG. 3A) showed that itwas derived from a functionally rearranged β gene composed of the Vβ5.1leader, Vβ2; a short portion of the Dβ2 segment, Jβ2.3; and the Cβ2 genesegment. One of the two β alleles, isolated in clones λ 8 by restrictionmapping and DNA sequencing showed that it contains the rearranged Vβ8.2gene about 100 bp downstream of an apparently inactive Vβ8.2 leaderexon, while the Vβ5.1 leader sequence is located about 2.5 kb fartherupstream (FIG. 3B). Splicing of the Vβ5.1 leader exon to the Vβ8.2 exonin T cells derived from C57BL/6 mice has been described by Chou, et al.(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1992-1996. The second β allelein B6.2.16 contains an incomplete Dβ1-Jβ2.5 rearrangement (not shown).

For the production of transgenic mice, a functionally rearranged β genewith long 5' and 3' flanking sequences was used. This was becauseinitial experiments with clone λ8 containing only 1.2 kb of 5' and 3 kbof 3' flanking sequence failed to give expression in nine independentlyobtained transgenic mice.

Transgene Reconstitution

From the genomic clone λ9, a 13 kb SaII-SacII fragment, containing thefunctionally rearranged Vβ gene with 9 kb of 5' flanking sequence, wasfused by way of a unique Sac II site to a 23 kb Sac II-Sal I fragmentcontaining Jβ2 and Cβ2 gene segments together with 18 kb of 3' flankingsequence. The latter fragment was isolated from cosmid clone cos H-2^(d)II-1.14 T derived from a BALB/c liver library, previously described(Steinmetz, et al. (1986) Cell 44, 895-904). See FIG. 3. Both fragmentswere ligated in the presence of Sal I-digested pTCF cosmid vector DNAarms. Ligation was checked by agarose gel electrophoresis. In vitropackaging and transformation of E. coli strain 490A was carried out aspreviously described (Steinmetz, et al. (1985) Methods in MolecularImmunology in Immunological Methods, Vol. III, Letkovits and Pernis,eds. (Orlando, Fla., Academic Press)). Several clones were picked andclone cos HYβ9-1.14-5, containing the reconstituted transgene as shownin FIG. 3B, was identified by restriction endonuclease mapping.

Transgenic Mice

The 36 kb insert of cos HYβ9-1.14-5 was released by SaII digestion andisolated by preparative agarose gel electrophoresis and electroelution.The DNA was extracted twice with phenol-chloroform and precipitated withethanol. The DNA pellet was dissolved in ultrapure water and dialyzedagainst 10 mM Tris-HCl, 0.1 mM EDTA (pH 7.4). The DNA was adjusted to afinal concentration of 4 μg/ml. Fertilized mouse eggs were recovered incumulus from the oviducts of superovulated (CBA/BrA×C57BL/LiA)F₁ femalesthat had mated with F1 males several hours earlier. Strains CBA/BrA andC57BL/L.A were obtained from The Netherlands Cancer Institute breedingfacility, Amsterdam. Approximately 100 copies of the β gene wereinjected in the most accessible pronucleus of each fertilized egg, asdescribed by Hogan, et al. (1986) Manipulating the Mouse Embryo (ColdSpring Harbor, New York, Cold Spring Harbor Laboratory Press.Microinjected eggs were implanted into the oviducts of one-daypseudopregnant (CBA/BrA×C57BL/LiA)F₁ foster mothers and carried to term.Several weeks after birth, total genomic DNA was isolated from tailbiopsies of the pups. Mice that had incorporated the injected DNA intheir genome were used for further breeding.

Southern and Northern Blot Analyses

Tail DNA was purified from the terminal quarter of tails of 4-week-oldmice. The skin was separated from the bone and homogenized in 1 ml of 1%NaCl, 10 mM EDTA (pH 8.0), on ice by using a Polytron with a PTA7 blade.DNA was isolated by phenol-chloroform extraction and dialyzed against 10mM Tris, 1 mM EDTA (pH 8.0) (TE). One milliliter of 6% p-aminosalicylatewas added to the first phenol extraction. Mouse organs were chopped inice-cold PBS and homogenized in 4M guanidinium isothiocyanate. 0.5 mMsodium citrate (pH 7.0), 0.1 M β-mercaptoethanol, 0.5% Sarkosyl, using aPolytron as above. From the homogenate, RNA and DNA were isolated bydifferential centrifugation as described previously (Maniatis, et al.(1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.,Cold Spring Harbor Laboratory Press). DNA-containing fractions weredialyzed against TE buffer for 6 hr. extracted with phenol-chloroformthree times, and dialyzed against TE overnight. Total cellular RNA (5 to10 μg) was separated on 1.5% formaldehyde-agarose gels according toRave, et al. (1979) Bucl. Acad. Res. 6, 3559-3527) and transferred toZeta probe membranes (BioRad) or BA85 nitrocellulose filters (Schleicher& Schull). Genomic DNAs (10 μg) were digested to completion withrestriction endonuclease, separated on 0.6% agarose gels, andtransferred to Zeta probe membranes according to Reed and Mann (1985)Nucl. Acad. Res. 13, 7201-7221.

Northern blot hybridization with labeled restriction fragments wasperformed by using the same conditions as for Southern blothybridization described by Steinmetz (1986), supra. Hybridization withthe 30-mer oligonucleotide containing the VDJ joining region of thetransgene was done in 900 mM NaCl, 90 mM Tris-HCI (pH 7.2), 6 mM EDTA,10× Denhardt's solution, 1% SDS, and 500 μg/ml E. coli DNA at 42° C.overnight. After hybridization the filter was washed twice in 6× SSC,0.1% SDS, at room temperature and twice in 3× SSC, 0.1% SDS, at 60° C.for 10 min. each. Southern blot hybridization was carried out asdescribed by Uematsu, et al. (1988) Immunogenetics 27, 96-101. Filterswere exposed with Kodak X-Omat S or X-Omat AR films at -70° C. for 2 hr.to 1 month using intensifying screens.

For hybridization we used previously described Cα and Cβ probes wereused (Dembic, et al. (1985) Nature 314, 271-273; Snodgrass, et al.(1985) Nature 313, 592-595. The Jβ2 probe is a 1.2 kb CIaI-EcoRIfragment located immediately downstream of the Jβ2 gene segments (Chien,et al. (1984) Nature 309, 322-326). The Dβ1 probe is a 1.4 kb upstreamof the Dβ2 gene segment (Siu, et al. (1984) supra. The Cβ1-specificprobe was derived from the 3' untranslated region as described byGascoigne, et al. (1984) Nature 310, 387-391. The transgene-specific VDJprobe is an oligonucleotide of 30 residues in length identical to thecomplementary strand of the underlined cDNA sequence shown in FIG. 3Afrom position 581 to 610. Vβ7, Vβ9, Vβ11, Vβ12, Vβ14, Vβ15 and Vβ16probes have been described by Lai, et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 81, 3846-3850.

Surface Staining of Lymphocytes

Single-cell suspensions were prepared from lymph node or spleen. Cellswere incubated with various monoclonal antibodies purified fromsupernatants of antibody-producing hybridoma cells. Cells (106) wereincubated with a saturating dose of antibody at 0° C. for 20 min,washed, and incubated again with fluorescein isothiocyanate (FITC)coupled goat anti-mouse imnunoglobulin antibodies. In some experiments,antibodies coupled with biotin were used in the first step followed byincubation with FITC-avidine (Kisielow, et al. (1984) J. Immunol. 133,1117-1123).

Modulation and Immunoprecipitation of Surface Antigens

For modulation, cells were incubated with an excess of F23.1 antibodies(50 μg/ml) and rabbit anti-mouse immunoglobulin antibodies for 24 hr. at37° C. Viable cells were obtained by spinning the suspension throughFicoll. For immunoprecipitation, 10⁷ B6.2.16 cloned T cells, C57BL/6 Tcells, and 93.2 T cells were labeled with 0.5 mCi of NaI (Amersham) bythe lactoperoxidase/glucoseoxidase method (Goding (1980) J. Immunol.124, 2082-2088) for 20 min. at room temperature in PBS. The cells werewashed five times with ice-cold PBS containing 0.1% NaN₃ and 17 mg/mlKCI and were then lysed for 30 min. on ice with a buffer containing 2%Triton X-100, 20 mM Tris (pH 8), 150 mM NaCl, 3 mM MgCl, 0.1 mM PMSF,and 5 mM iodoacetamide. The lysate was centrifuged for 5 min. in anEppendorf centrifuge. Half of each sample was precleared four times with100 μl of F23.1 antibodies coupled to Sepharose beads. Precleared anduntreated lysates were then incubated with 5 μg of an anti-β panspecificantibody (Traunecker, et al. (1986) Eur. J. Immunol. 16, 851-854) for 2hr. on ice, and subsequently incubated with 35 μl of 50% protein Abeads. All samples were washed with high sale (lysis buffer containing 1mg/ml ovalbumin and 0.65 M NaCl) and low salt (lysis buffer containing0.15 M NaCl) buffers. Samples were reduced with 2-mercaptoethanol andanalyzed on a 12.5% polyacrylamide gel. Autoradiography was accomplishedwith Kodak X-Omat AR film and intensifying screens.

Generation of Cytolytic T cells and Cytolytic Assay

Spleen (10⁷) or lymph node (10⁷) cells were cultured with 10⁷x-irradiated (2000 rads) allogeneic stimulator cells for 5 days in 4 mlof culture medium. ⁵¹ Cr-labeled target cells were prepared bystimulating spleen cells from various mouse strains with concanavalin A(Con A) (2.5 μg/ml) for 48 hr. at 10⁶ cells per ml. Con A blasts werepurified by centrifugation over Ficoll, and viable cells were ⁵¹Cr-labeled by incubation in ⁵¹ Cr for 1 hr. at 37° C. In the cytolyticassay, 10⁴ 51 Cr-labeled targets were incubated with various numbers ofcytolytic T cells for 3.5 hr. at 37° C. in serial bottom wells in 200 μlof medium. The plates were centrifuged and 100 ml of supernatant removedto determine released ⁵¹ Cr (Pohlit, et al. (1979) Eur. J. Immunol. 9,681-690). For blocking of activity, cytolytic T cells were incubated for30 min. at 37° C. with F23.1 antibodies (50 μg/ml). Then ⁵¹ Cr-labeledtargets were added and the cytolytic assay were performed as describedabove.

Establishment of Cell Lines and T Cell Clones from Transgenic Mice

Cell lines were made by stimulation of spleen cells with either Con A orallogeneic x-irradiated stimulator cells in medium containing IL-2.After 10 to 14 days, the cells were washed and restimulated (10⁶responder cells, 10⁷ x-irradiated stimulator cells) in IL-2-containingmedium. Viable cells were obtained by centrifugation over Ficoll.Cloning was carried out by seeding 0.3 cells per well in 96-wellmicrotiter plates containing 10⁶ X-irradiated feeder spleen cells perwell and 200 μl of IL-2-containing medium. After 1 to 2 weeks, growingcolonies were transferred together with 10⁷ x-irradiated feeder cellsinto 2 ml of IL-2-containing medium in 24-well Costar plates. From thenon, restimulations were carried out at 7-day intervals with 10⁶ clonedcells and 10 x-irradiated stimulators per 1 ml of IL-2-containingculture medium.

Transgenic Mice Express the Introduced T Cell Receptor β Gene

Thirteen pups were born that contained from 1 to about 50 copies of thetransgene, according to Southern blot analysis of tail DNA. Of thesemice, seven were analyzed after splenectomy for transcription with theoligonucleotide probe that covered the VDJ joining region and that wastherefore specific for the transgene. Six of the seven mice showed a 1.3kb full-length transcript. As observed before for other transgenes, nosimple correlation between copy number and transcriptional activity wasseen. T lymphocytes obtained from lymph nodes of four transgenic micewere subsequently analyzed for cell-surface expression of the transgenicβ chain on a fluorescence-activated cellsorter (FACS) by using F23.1antibodies (Table 1). While about 10%-20% of T cells were F23.1-positivein mice 90 and 91, practically all of the T cells from mice 93 and 95were stained with the F23.1 antibody. This indicates that most, if notall, of the T cells in mice 93 and 95 express the transgenic β chain onthe cell surface. Since a background of about 10%-20% F23.1-positive Tlymphocytes is expected for these mouse strains (Staers, et al. (1985)J. Immunol. 134, 3994-4000), it is unlikely that the transgenic β chainis expressed in mice 90 and 91. In agreement with the FACS analysis,Northern blot analysis did not show any transgene transcription in Tlymphocytes of mouse 90 (Table 1).

                  TABLE 1                                                         ______________________________________                                        Transgenic Mice Express the                                                     Introduced T Cell Receptor β Gene                                                                       Transgene                                                                            T Cell                                   Off-  Copy Tran- Surface                                                     Founder spring Sex.sup.a Number.sup.b scription Expression.sup.c            ______________________________________                                        90             m       1       -      10%-20%                                   91  f 20 ND 10%-20%                                                           92  f 10 ND ND                                                                93  f 5 + >98%                                                                 93.1 m 0 ND ND                                                                93.2 m 20 + >98%                                                              93.4 m 2 + >98%                                                               93.9 f 20 + >98%                                                             94  m 3 + ND                                                                  95  f 2 + >98%                                                                 95.39 m 2 + ND                                                                95.40 m 2 ND ND                                                            ______________________________________                                         .sup.a Sex is indicated by m (male) and f (female).                           .sup.b Copy number of the transgene was estimated from signal intensities     on Southern blots with Pvulldigested tail DNA bybridized with the Jβ     probe.                                                                        .sup.c Surface expression of F23.l positive β chains on lymph node T     cells was quantitated by FACS analysis.                                       ND  Not determined.                                                      

The male founder mouse 93 was subsequently crossed with female C57L mice(a F23.1-negative mouse strain; Behlke, et al. (1986) Proc. Natl. Acad.Sci. U.S.A. 83, 767-771. Eighteen offspring mice from the firstgeneration were aalyzed for the inheritance of the transgene by Southernblot hybridization. Three (1 male, 2 females) contained about 20 copiesof the transgene, 5 (4 males, 1 female) contained about 2 copies, and 10lacked the transgene (FIG. 4). This blot shows the anaysis of 9 mice. Itdemonstrates the inheritance of the transgene (indicated by the 7.7 kbband) in either 2 (mice 93.3, 93.4, 93.7) or 20 (93.2, 93.9) copies. The5.5 kb band represents the endogenous β locus. Southern blot analysis ofPvull-digested tail DNA was carried out as described with a Jβ2 probe.The segregation of the transgene into 2 and 20 copies could be due tointegration of injected DNA into two unlinked chromosomal loci, or couldreflect a deletion or amplification event after integration at a singlesite. Analysis of second generation offspring mice shows that the twodifferent forms of the transgene are stably inherited. The transgene istherefore present in germ-line DNA and transmitted in a Mendelianfashion to both makes and females. Mice with 2 or 20 copies of thetransgene were analyzed for cell-surface expression with F23.1antibodies. Lymph note T cells stimulated in vitro by irradiatedallogeneic DBA/2 spleen cells were incubated with Vβ8-specific F23.1antibodies followed by FITC-labeled sheep anti-mouse immunoglobulinantibodies, and were analyzed on FACS. In parallel, the expression ofCD3 molecules was monitored by using the 145.2C11 monoclonal antibody(Leo, et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1374-137??)followed by FITC-labeled goat anti-hamster immunologlobulin antibody. Asshown in FIG. 5, most, if not all, of the peripheral T lymphocytesanalyzed in mice 93.2 (20 copies) and 93.4 (2 copies) stain with theVβ8-specific monoclonal antipody. The staining intensity is similar inboth T lymphocyte populations, indicating that despite the difference incopy number, these mice express the transgene to the same extent.

Lymph node cells from mice with 2 copies of the transgene were analyzedfor the ratio of CD4- and CD8-positive T cells. Lymph node cells from atransgenic male mouse were 45% CD4⁻ and 18% CD8⁺, and from a transgenicfemale mouse, 35% CD4⁺ and 29% CD8⁺ ; control cells from a C57BL/6 mousewere 45% CD4 and 32% CD8⁺. These numbers do not deviate significantlyfrom those normally seen. Thus, although the transgenic β chain, derivedfrom a class I-restricted cytotoxic T cell clone, is expressed in most,if not all, of the T lymphocytes, it does not affect the normal ratio ofCD4- and CD8-positive T cells.

Transcription of the Transgene is Largely T Cell-Specific

Mouse 93.2, containing 20 copies of transgene was analyzed fortissue-specific expression of the transgene. Northern blot analysisusing the VDJ oligonucleotide as a probe reveals strong transcription ofthe thymus, while little or not transcription is seen in the othertissues analyzed (FIG. 6A). Similar results were obtained for transgenicmice 93.4 and 95.39, containing 2 copies of the transgene (not shown).The small amounts of transcripts we see in some nonlymphoid tissuescould be due to infiltrating T lymphocytes or incomplete inactivation ofthe transgene. N lymphocytes also show a low level of transgenetranscription. Both the VDJ oligonucleotide and the Cβ probe identify βchain transcripts in B lymphocytes, while α chain transcripts, evenafter a longer exposure, are not seen (FIG. 6B). The observedtranscripts of the transgene are therefore not due to residual T cellcontamination of the B-cell preparation, but reflect an about 20-foldlower level of transgene transcription in B as compared to Tlymphocytes.

Transgenic T Lymphocytes Express Only F23.1-Positive β Chains, WhichParticipate in the Formation of Functional Receptors

The F23.1 staining experiments indicate that the transgene is expressedin the vast majority of T lymphocytes. It is possible that additional βchains are encoded by endogenous genes. Several independent approacheswere used to check this possibility. In a first kind of experiment,membrane extracts from T cells of transgenic mice were "precleared" ofVβ8 proteins by using F23.1 antibodies coupled to Sepharose beads. Asshown in FIG. 7, after preclearing, no further β chains wereprecipitated by a panspecific antiserum (Traunecker, et al. (1986) Eur.J. Immunol. 16, 851-854) for mouse β chains. When the originalmale-specific clone B6.2.16 and T cells from C57BL/6 mice were analyzedfor controls, the panspecific antiserum precipitated β chains from thealtter but not from the former cells after preclearing with F23.1antibodies. In a second kind of experiment, T cell receptors weremodulated (by capping and endocytosis) by F23.1 antibodies and rabbitanti-mouse immunoglobulin at 37° C. Modulated and nonmodulated cellswere then stained with anti-CD3 antibodies (Leo, et al. (1987) supra.).As shown in FIG. 8, modulation with F23.1 antibodies removed all T cellreceptor-associated CD3 molecules from cell surfaces of the transgenicmice and from the B6.2.16 clone but not from other T cells. A third kindof experiment showed that the activity of cytolytic T cells fromtransgenic mice on allogeneic DBA/2 and C3H/HeJ target cells wascompletely blocked by F23.1 antibodies, whereas those from normalC57BL/6 mice were not inhibited significantly when used on the sametargets (FIG. 9). Lysis of allogeneic target cells after in vitrostimulation was specific. Cytolytic T cells from transgenic micestimulated with DBA/2 cells lysed DBA/2 but not C3H/HeJ target cells,and stimulation with C3H/HeJ cells gave rise to C3H/HeJ-directed, butnot DBA/2-directed, killer cells (not shown). Taken together, theseexperiments indicate that only β chains encoded by the transgene areexpressed o T lymphocytes of the transgenic mice and that they are usedto form functional T cell receptors.

Endogenouse β Genese are Incompletely Rearranged

Rearrangement of endogenouse β genes can be distinguished from therearranged transgene by using specific hybridization probes. With a Cβprobe, rearrangements of the Cβ1 locus can be separated from those atthe Cβ2 locus (which is present in the transgene) if HindIII-digested Tcell DNa is analyzed. This analysis clearly shows that the 9.4 kbgerm-line HindIII fragment containing the Cβ1 locus is rearranged intransgenic T lymphocytes from mouse 93.2 (FIG. 10A). The rearrangements,however, appear to be qualitatively different from those seen in normalC57L T lymphocytes used as a control. While no discrete rearrangedfragments appear in C57L T cell DNA, a series of fragments (not seen ina B cell control) is evident in T cell and Thymus DNA from trnsgenicmouse 93.2. Also, mouse 93.1, which did not inherit the transgene, lookslike the C57L control.

With a probe derived from the 5' flanking region of the Dβ1 genesegment, a series of discrete rearranged fragements is again seen intransgenic but not in control T cell, nor in Transgenic B cell, DNA(FIG. 10B). This result shows that partial Dβ1 rearrangements,presumably to Jβ1 and Jβ2 gene segments, are present in unusually highfrequencies in the transgenic mouse. Indeed, the sizes of the observedrearranged fragments are in agreement with this assumption. Since thesefragments are identified with a 5' flanking probe of Dβ1, they representpartial rearrangements that do not involve Vβ gene segments. Inversionof the Vβ14 gene segment, located downstream of Cβ, would not delete the5' flanking sequence of Dβ gene segments (Malissen, et al. (1986) Nature319. 28-33). Analysis of T cell clones from transgenic mice (see below)with a Vβ14 probe, however, shows that endogenouse Vβ14 genese have notbeen rearranged to Dβ gene segments. In agreement with the Southern blotanalysis, a Northern blot analysis of thymus and peripheral T cell RNAwith a mixture of ³² P-labeled Vβ7, Vβ9, Vβ11, Vβ12, Vβ14, Vβ15 and Vβ16gene segments did not reveal Vβ gene transcripts in transgenic mouse93.2, while a clear signal was obtained with a C57BL/6 control (notshown).

In contrast to the Dβ1 gene segment, the Dβ2 gene segment does notundergo frequent rearrangements in transgenic mouse 93.2. As shown inFIG. 10C, it rather seems that most of the Dβ2 gene segments remain ingerm-line configuration in 93.2 thymocytes and T cells. Mouse 93.4,containing only 2 rather than 20 copies of the transgene, shows asimilar high frequency of partial Dβ1 rearrangements (not shown).Furthermore, mouse 95.39, independently generated, and a transgenicmouse obtained by coinjection of the same β gene together with a T cellreceptor α0 gene show a predominance of partial Dβ1 rearrangements (notshown). Thus neither the high coy number of the transgene in mouse 93.2not its peculiar shromosomal location causes this unusual pattern ofrearrangement.

The results presented so far do not exclude a low but still significantnumber of complete endogenous VDJ rearrangements. To obtain a morequantitative estimate, we analyzed nine T cell clones obtained from mice93.2 and 93.4 with the same Cβ, Dβ1, and Dβ2 probes. Of the 18endogenouse β loci screened, 13 rearranged Dβ1 to Jβ1, 2 fused Dβ1 withJβ2, 3 joined Dβ2 with Jβ2, and 2 show unusual rearrangements of bothDβ1 and Dβ2 gene segments that were not further characterized (Table 2).No complete VDJ rearrangements were found, indicating that they occurvery rarely if at all.

                  TABLE 2                                                         ______________________________________                                        T Cell Clones Analyzed                                                                    Rearrangements.sup.a                                                              Dβ1                                                                              Dβ1 Dβ2                                                                            Dβ1                                 Mouse Clone.sup.b to Jβ1 to Jβ2 to Jβ1 to Jβ1.sup.c     ______________________________________                                        93.2   2        1                1     1                                          8 1 1                                                                        14 1   1                                                                      16 .sup. 2.sup.d                                                              17 2                                                                          18 1 1                                                                        19 2                                                                           20.sup.e 1  2                                                               93.4  2 2                                                                   ______________________________________                                         Notes:                                                                        .sup.a Rearrangements of the two homologous loci in each T cell clone wer     analyzed with Cβ, Dβ1, and Dβ2 probes.                         .sup.b T cell clones were obtained as described in Experimental               Procedures.                                                                   .sup.c The sizes of these rearranged fragments do not correspond to those     expected for DJ rearrangements.                                               .sup.d Indicates that DJ rearrangements have occured on both homologous       chromosomes.                                                                  .sup.e Clone 20 contains one β locus in germline configuration.     

Expression of the Transgene Regulates Rearrangement of Endogenous βGenes

The majority of endogenous β genes in the transgenic mice showrearrangements of Dβ1 to Jβ1 gene segments. No endogenous VDJrearrangements are seen. The preponderanve of incomplete Dβ1 to Jβ1rearrangements is highly unusual, and is independent of the copy numberof the transgene and its chromosomal location. It can be deduced fromthe analysis of T cells and T cell clones that approximately half of thenonproductive β rearrangements in normal T cells represent VDJrearrangements. Of eight nonfunctional alleles that have beencharacterized in T cell clones, five show partial DJ rearrangements (atleast three of which are Dβ1-Jβ2) and three show aberrant VDJrearrangements. Expression of the introduced βgene in transgenic micetherefore blocks endogenous β gene rearrangement between D-J and V-DJjoining.

EXAMPLE 2 T Cell Depleted Transgenic Mice

Transgenic nice depleted in T cell population were produced byintroducing a transgene encoding the functionally rearranged T cell βreceptor chain of Example 1 wherein approximately 90% of the variableregion of the β receptor gene was deleted.

Transgene Constructions

The 20 kbp KpnI fragment derived from cosHYβ9-1.14-5 (FIG. 3B) wassubcloned in PUC18. This plasmid was deposited with the CentraalbureauVoor Schimmelcultures (CBS) Baarn, The Netherlands on Dec. 5, 1988 andassigned deposit No. CB5726.88. The construction of the deletiontransgenes from this plasmid is illustrated schematically in FIGS. 11A,11B and 12. The functional TCR β gene from the B6.2.16 contains inaddition to the fused Vβ8.2, Dβ2 and Jβ2.3 segments the J segmentsJβ2.4, Jβ2y and Jβ2.6. A 4.0 kb XbaI fragment contains the VDJ join andthe remaining J segments. This fragment was inserted into pSP64(Promega, Madison, Wis. to form pSP4X). In this region of the KpnIfragment three PstI sites are present, the first at 17 bp from the startof the Vβ8.2 segment, the second between the Jβ2.5 and the Jβ2ψ and thethird at 22 bp downstream from the Jβ2ψ segment. The 707 bp locatedbetween the first and the last PstI sites were deleted from the XpnIfragment thereby removing all but the first six codons of the Vβ8.2segment, Dβ2 and Jβ2-Jβ5. Two complementary oligonucleotide sequenceswere synthesized corresponding to the last six codons of the Jβ2.3segment and the 34 bp region downstream from the Jβ2.3 segment includingthe splice donor sequences. The oligomers were prepared in such a waythat a PstI site is generated a the border in order to facilitateinsertion into a PstI site. Digestion of pSP4X and religation resultedin the pSP2.7XP plasmid carrying a 2.7 kbp XbaI/PstI fragment in whichall of the Vβ8.2 sequences, except for the first 17 bp, and the 3'sequences up to the Xba site were deleted. pSP2.7XP was cleaved withPstI and religated in the presence of excess of reannealed oligomers(for sequence data see FIG. 11). The sequence chosen at the 5' and 3' ofthe oligomers was chosen such, that insertion of reannealed oligomer inthe correct orientation restores only the 3' PstI restriction site.Plasmid pSP2.7XP contained one copy of the synthetic of 57 mer in thecorrect orientation. In the PstI site of pSP2.7XP the 550 bp PstIfragment containing the Jβ2.6 segment derived from the pSP4X subclonewas inserted to yield the pSP3.2X* clone. pSP3.2X* carries a 3.2 kb XbaIfragment identical to 4.0 kbp XbaI insert of the pSP4X clone except forthe deletion of the 707 bp PstI fragment. Finally, the 4.0 kb XbaIfragment in the 20 kb KpnI fragment was replaced by the 3.2 kb XbaIinsert of pSP3.2X* via multiple cloning steps involving the unique SacIIsite. Such multiple cloning steps are shown in FIG. 12. This resulted inthe formation of the ΔV-TCRβ λ₀νε.

A ΔV.sub.▪ -TCRβ clone containing one extra nucleotide was obtained inthe same way, except that the pSP2.7XP subclone incorporated oligomersthat contained the extra nucleotide in the coding region (FIG. 11).Because of the presence of the additional nucleotide the mRNAtranscribed from this construct can not be translated into a polypeptidecarrying a C region. This construct served as a control afterintroduction into transgenic mice. All manipulations involving thedeletion of the 707 bp PstI fragment and the insertion of the oligomerswere checked by nucleotide sequence analysis.

Generation of Transgenic Mice

The DNA fragments (ΔV-TCRβ or ΔV.sub.▪ -TCRβ) that were used forinjection were released from the above vectors using the appropriaterestriction endonucleases and purified as described earlier(Krimpenfort, et al. (1988) Embo. J. 7, 745). The final DNAconcentration was adjusted to 2 μg/ml. Fertilized eggs were recovered incumulus from the oviducts of superovulated (CBA/BrA×C57Bl/LiA) F1females that had mated with F1 males several hours earlier. The DNAfragments were injected into the most accessible pronucleus of eachfertilized egg essentially as described (Hogan, B. J. M., et al. (1985)Manipulation of the Mouse Embryo: A Laboratory Manual, Cold SpringsHarbor Laboratory Press, Cold Spring Harbor, N.Y.). After overnightculturing two-cell-stage embryos were implanted into the oviducts ofpseudopregnant fosters (F1 or BDF) and carried to term. Several weeksafter birth total genomic DNA was prepared from tail biopsies asdescribed (Hogan, et al., supra)

DNA Analysis

For Southern blot analysis, 8 μg total genomic DNA was digested withrestriction endonucleases as recommended by the supplier, separated onagarose gels and transferred to nitrocellulose. Filters were hybridizedto 32P-labeled probes as described (Cuypers, H. T., et al. (1984) Cell37, 141). Final wash was at 0.1× SSC, 42° C. The probe used for thetransgene analysis was an EcoRI/HincII fragment located downstream fromthe Jβ2 cluster. This probe recognizes a 6.0 kbp PvuII fragment ingermline DNA, whereas a 7.9 kbp PvuII fragment was diagnostic for the ΔVconstructs.

RNA Analysis

5-15 μg of total RNA, prepared by the LiCl-urea method (Auffray, C., etal. (1980) Eur. J. Biochem. 177, 303) was separated on 1% agaroseformaldehyde gels (Maniatis, et al. (1982), supra.) and transferred tonitrocellulose. Probes used for RNA analysis were Cβ probe comprising acDNA fragment representing the Cβ2 region which also hybridizes with Cβ1transcripts (Snodgrass, H., et al. (1985) Nature 313, 592) and an actinprobe (Dodemont, H. J., et al. (1982) EMBO J. 1, 167). These probes were32P-labeled by nick-translation. Hybridization conditions were asdescribed (Cuypers, et al. (1984) Cell 37, 141). Final wash was at 0.1 ×SSC and 60° C.

FACS Analysis

Analysis was performed on a FACS II analyzer (Becton and Dickinson)using established protocols (Scollay, R., et al. (1983) Thymus 5, 245).Single cell suspensions were prepared from spleen in balanced saltsolution containing 5% fetal calf serum and 0.5 mg/ml sodium azide.Cells were stained with saturating amounts of antibodies andcounterstained with FITC-coupled rabbit anti-rat antibodies (Vesmel, W.,et al. (1987) Leukemia 1 155). The origin and specificity of theantibodies were as follows: L3T4 (monoclonal 129-19 (Pierres, A., et al.(1984) J. Immunol. 132, 2775), Thy-1 (monoclonal SgA D2-2), Lyt-2(monoclonal 53-6-7).

Transgenic Mice

Two transgenic founders were obtained carrying the ΔV.sub.▪ -TCRβconstruct. In both transgenic mice multiple copies had integrated inhead-to-tail configuration (data not shown). RNA was isolated fromspleen of these mice to check transcription of the ΔV.sub.▪ -TCRβtransgene (see below). One ΔV.sub.▪ -TCRβ founder was sacrificed forobduction. No abnormalities were observed (normal thymus, spleen).

Using the ΔV-TCRβ construct eight transgenic mice were generated. Inmost of these mice multiple copies had integrated. Mouse #1670 (male)did not produce offspring and was sacrificed for analysis. Strikingly,the thymus in this founder was strongly reduced in size. Noabnormalities were recorded in other tissues. All transgenic offspringfrom another transgenic mouse (#1733) had an even more pronouncedphenotype as mouse #1670: no thymus was present, whereas other tissuesappeared normal. No abnormalities were observed in negative offspringfrom this transgenic line. Splenocytes from founder #1670 and frompositive offspring from #1733 were analyzed by FACS.

Transgene Transcription

As discussed above, the ΔV.sub.▪ -TCRβ differs in only one nucleotidefrom the ΔV-TCRβ transcripts and therefore does not encode a polypeptidechain containing a TCR constant region.

Two RNA species are transcribed from TCRβ genes, 1.0 kb and 1.3 kb insize. Partially (DJ) rearranged β genes yield the 1.0 kb transcripts,whereas the fully (VDJ) rearranged β genes give rise to the 1.3 kb RNAs.In thymus both TCRβ transcripts are present abundantly (Snodgrass, etal. (1985) supra.). In spleen, however, the 1.0 kb TCRβ transcript ishardly detectable. Correct transcription and processing of the ΔV geneconstructs also lead to a 1.0 kb mRNA. Northern blot analysis usingprobes specific for the Cβ region cannot discriminate between ΔV.sub.▪-TCRβ transcripts and DJ transcripts. Therefore, RNA from spleen ratherthan thymus was chosen for the analysis of the ΔV.sub.▪ -TCRβ transgeneexpression. RNA was isolated from spleen, from control mice, and frommice carrying the ΔV.sub.▪ -TCRβ construct. As can be concluded formFIG. 13 the endogenous TCRβ gene expression is not affected in theΔV.sub.▪ -TCRβ transgenic mice. In spleen from control and transgenicmice a 1.3 kb band of approximately equal intensity can be detected withthe Cβ probe. This indicates that complete VDJ rearrangements are notinhibited in the ΔV.sub.▪ -TCRβ transgenic mice. However, RNA from thetransgenic spleens shows a high expression of a 1.0 kb RNA species thatis probably derived from the transgene ΔV.sub.▪ -TCRβ. The difference inexpression level between the two transgenic strains supports thisinterpretation. We conclude from the RNA analysis, that the ΔV.sub.▪-TCRβ and therefor probably also the ΔV-TCRβ transgenes give rise tostable transcripts.

Results of FACS Analysis

As it was impossible to isolate a sufficient number o cells from thethymus of the transgenic mice carrying the ΔV-TCRβ construct, FACSanalysis was only performed on splenocytes. Splenocytes were isolatedfrom founder #1670 and from transgenic progeny of lines #1673 and #1733.As a control cell suspensions were prepared from the spleen of negativelittermates. Splenocytes were stained with anti-sera specific for the Bcell marker surface immunoglobylin and the T cell markers Thy-1, L3T4(T4) and Lyt-2 (T8). The FACS data on cell suspensions from differenttransgenic mouse lines were essentially the same. In the control cellsuspension the B cell specific anti-sera show a reactive and anunrective cell population, representing B and T cells, respectively(FIG. 14). In contrast, in the transgenic cell preparation (1670) allcells stain with the B cell specific anti-sera. This indicates, that allcells in the transgenic spleens are of B cell origin. This observationis supported by the staining patterns obtained with the T cell specificanti-sera. Whereas in the control spleen approximately 30% of the cellscan be stained with the Thy-1 anti-serum, only a minor fraction if anyof the transgenic splenocytes are Thy-1 positive. The same observationswere made by FACS using the L3T4 and Lyt-2 anti-sera. Apparently, Tcells are absent form the spleen of the ΔV-TCRβ transgenic mice.

Conclusion

The RNA analysis of spleen of transgenic mice carrying the ΔV.sub.▪-TCRβ gene construct shows that the introduced ΔV constructs are highlyexpressed. Moreover, the ΔV.sub.▪ -TCRβ transgenic mice show, that thetranscriptional activity of the ΔV constructs per se does not inhibitthe complete rearrangements of the endogenous β chain genes, since inthe ΔV.sub.▪ -TCRβ transgenic mice the 1.3 kb VDJ is expressed atsimilar levels as in control mice. The analysis of the ΔV-TCRβtransgenic mice is still preliminary, e.g. at the moment no data on γδbearing T cells are available. A few consistent observatons were made:no or only a rudimentary thymic structure was left in several ofindependent ΔV-TCRβ transgenic mice lines; FACS analysis on splenocytesfrom these mice demonstrated that T cells were virtually absent. Theabsence of T cells in the ΔV-TCRβ transgenic mice can be explained inseveral ways. Firstly, the ΔV transgenic protein itself might be lethalfor T cells. This is unlikely, since a high expression of thestucturally similar Dμ-protein is not harmful for A-MuLV transformedpre-B cells. Moreover, it was shown that pre-B cells with a highDμ-protein expression behave just like B cells that express a completeIg heavy chain: they proceed in the B cell differentiation pathway withrearrangements of the light chain genes (Reth, et al. (1985) supra.).Secondy, the mutant chain might interfere with T cell maturation. It hasbeen suggested that during thymic maturation T cells are subject to bothpositive and negative selection processes. These include the stimulationof thymocytes that are able to recognize foreign antigen in associationwith self MHC as well as the elimination of self-reactive cells. Thisselection requires the interaction with MHC determinants. TCRs play anessential role in these interactions. In ΔV-TCRβ trnsgenic mice themutant TCRβ probably prevents expression of the endogenous β chain genesby inhibiting the complete VDJ rearrangements in the same way as the Dμprotein blocks further rearrangements of the Ig heavy chain genes inA-MuLV transformed pre-B cells. Consequently, T cells in these mice donot express a complete β chain gene and therefore do not carry a TCR,that can functionally interact with MHC determinants. As a result all Tcells die. Three mechanisms could account for the actual loss of Tcells: (i) active deletion of T cells that bear a non-functional TCR;(ii) the deleted TCR transgene forms a receptor complex that isselfreactive and therefore is clonally deleted; (iii) T cells of theΔV-TCRβ transgenic mice are unable to respond to proliferative stimuli.The available data do not discriminate between these alternatives.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

What is claimed is:
 1. A transgenic mouse lacking endogenous heavy chainJ segments and having a phenotype characterized by an absence of plasmaB cells producing naturally occurring mouse antibodies, the phenotypebeing conferred by an immunoglobulin heavy chain targeting transgeneintroduced into an ES target cell and integrated into somatic and germcells of the transgenic mouse or an ancestor thereof by homologousrecombination with a cognate endogenous immunoglobulin heavy chainallele to yield a targeted imnunoglobulin heavy chain allele whichcomprises a deletion of said J segments, the immunoglobulin heavy chaintargeting transgene comprising DNA sequences for identification andselection of ES cells containing the transgene in the targeted alleleand DNA sequences of a mouse heavy chain immunoglobulin gene havingsufficient sequence homology to recombine with the endogenous allele insaid ES target cell.